1. Field of the Invention
The present invention relates generally to the fields of alginate production in Pseudomonas aeruginosa. More particularly, the present invention is directed to the algU gene, its protein product AlgU, and their use for the identification of agents that inhibit the interaction between sigma factors and the RNA polymerase complex, and in particular the RNA polymerase holoenzyme.
2. Description of the Related Art
Cystic Fibrosis (CF) is the most common inheritable lethal disease among caucasians. There are approximately 25,000 CF patients in the U.S.A. The frequency of CF in also remarkably high in several other countries (e.g., Canada, United Kingdom, Denmark), ranging from 1 in 400 to 1 in 1,600 live births. There are numerous CF centers in the U.S.A. and Europe--specialized clinical facilities for diagnosing and treating children and adolescents with CF.
Chronic respiratory infections caused by mucoid Pseudomonas aeruginosa are the leading cause of high morbidity and mortality in CF. The initially colonizing P. aeruginosa strains are nonmucoid but in the CF lung they inevitably convert into the mucoid form. The mucoid coating composed of the exopolysaccharide alginate leads to the inability of patients to clear the infection, even under aggressive antibiotic therapies. The emergence of the mucoid form of P. aeruginosa is associated with further disease deterioration and poor prognosis.
The microcolony mode of growth of P. aeruginosa, embedded in exopolysaccharide biofilms in the lungs of CF patients (Costerton et al., 1983), among other functions, also plays a key role in hindering effective opsonization and phagocytosis of P. aeruginosa cells (Pier et al., 1987; Pier 1992). Although CF patients can produce opsonic antibodies against P. aeruginosa antigens, in most cases phagocytic cells cannot effectively interact with such opsonins (Pressler et al., 1992; Pier et al., 1990; Pier 1992). Physical hindrance caused by the exopolysaccharide alginate and a functionally important receptor-opsonin mismatch caused by chronic inflammation and proteolysis are contributing factors to these processes (Pedersen et al., 1990; Tosi et al., 1990; Pier, 1992). Under such circumstances, the ability of P. aeruginosa to produce alginate becomes a critical persistence factor in CF; consequently, selection for alginate overproducing (mucoid) strains predominates in the CF lung.
Synthesis of alginate and its regulation has been the object of numerous studies (Govan, 1988; Ohman et al., 1990; Deretic et al., 1991; May et al., 1991). It has been shown that several alginate biosynthetic genes form a cluster at 34 min of the chromosome (Darzins et al., 1985), and that the algD gene, encoding GDPmannose dehydrogenase, undergoes strong transcriptional activation in mucoid cells (Deretic et al., 1987; 1991). GDP mannose dehydrogenase catalyzes double oxidation of GDP mannose into its uronic acid, a reaction that channels sugar intermediates into alginate production.
The transcriptional activation of algD has become a benchmark for measuring molecular events controlling mucoidy (Deretic et al., 1991; Ohman et al., 1990; May et al., 1991). Studies of these processes have lead to the uncovering of several cis- and trans-acting elements controlling algD promoter activity including: (i) The algD promoter has been shown to consist of sequences unusually far upstream of the mRNA start site (Mohr et al., 1990). These sequences (termed RB1 and RB2), as well as a sequence closer to the mRNA start site (RB3) are needed for the full activation of algD (Mohr et al., 1990; 1991; 1992). (ii) AlgR, a response regulator from the superfamily of bacterial signal transduction systems (Deretic et al., 1989), binds to RB1, RB2, and RB3, and is absolutely required for high levels of algD transcription (Mohr et al., 1990; 1991; 1992). (iii) Another signal transduction factor, AlgB, also contributes to the expression of genes required for alginate synthesis (Wozniak and Ohman, 1991). (iv) The peculiar spatial organization of AlgR binding sites imposes steric requirements for the activation process. The conformation of the algD promoter appears to be affected by histone like proteins [e.g. Alg (H.sub.p 1) (Deretic et al., 1992) and possibly IHF (Mohr and Deretic, 1992)], and perhaps by other elements controlling nucleoid structure and DNA topology. (v) The algD promoter does not have a typical -35/-10 canonical sequence (Deretic et al., 1989). It has been proposed that RpoN may be the sigma factor transcribing this promoter; however, several independent studies have clearly ruled out its direct involvement (Mohr et al., 1990; Totten et al., 1990). The present inventors have cloned and characterized a new gene, algU, which plays a critical role in algD expression (Martin et al., 1993).
The algU gene encodes a polypeptide product that shows sequence and domain similarities to the alternative sigma factor Spo0H from Bacillus spp. (Dubnau et al., 1988). Spo0H, although dispensable for vegetative growth, is responsible for the initial events in the triggering of the major developmental processes in Bacillus subtilis, viz. sporulation and competence (Dubnau et al., 1988; Dubnau, 1991). These findings suggest that activation of alginate synthesis may represent a cell differentiation process participating in interconversions between planktonic organisms and biofilm embedded forms in natural environments (Martin et al., 1993; Costerton et al., 1987).
Inactivation of algU abrogates algD transcription and renders cells nonmucoid, further strengthening the notion that algU plays an essential role in the initiation of mRNA synthesis at algD (Martin et al., 1993). algU maps in the close vicinity of muc markers that have been demonstrated in genetic studies by Fyfe and Govan (1980) to cause the emergence of mucoid strains constitutively overproducing alginate. The mucoidy-causing property of muc mutations has been based on the ability of different muc alleles (e.g. muc-2, muc-22, and muc-25) to confer mucoidy in genetic crosses (Fyfe and Govan, 1980; 1983). The present application also describes the presence of additional genes immediately downstream of algU, termed mucA and mucB, that also play a role in the regulation of mucoidy.
The characterization of the genetic components that lead to P. aeruginosa mucoidy, in particular the algU transcriptional activator, provide a new platform for the identification and isolation of new antibiotic agents. These agents will target the interaction of the algU gene product and RNA polymerase. Such drugs will represent a new class of antibiotics, since current examples of RNA polymerase inhibitors (e.g. Rifampicin) act on the individual subunits of the polymerase complex.